This project is designed to investigate DNA methylation (5-methylcytosine formation) during DNA excision repair in human diploid fibroblasts damaged with chemical carcinogens and UV radiation. Data from our laboratory and other laboratories suggest that excision repair is a template-directed, error-correcting process which restores the linear integrity of the DNA, eliminates mutagenic lesions and increases cell survival; however, it is unclear whether or not the biochemical signals which regulate gene expression are re-established during the repair process. Recent studies have indicated that one such signal is the extent of DNA methylation and its distribution in the genome. We will examine the extent and time course of DNA methylation during DNA excision repair in human diploid fibroblasts damaged with ultraviolet radiation (UV) and chemical carcinogens. Carcinogens include N-acetoxy-2-acetylaminofluorene, 7-bromomethylbenz(a)-anthracene, benzo(a)pyrene 7,8-diol-9,10-oxide, methylnitrosourea and methylnitronitro-soguanidine. We plan to determine if DNA methylation occurs only in repair patches or also occurs in adjacent, flanking sequences. These studies are possible because of a new approach to isolating repair patches and adjacent regions developed by us. By labeling cells during repair with BrdUrd and using antibody to BrdUrd, we can selectively precipitate these regions of the genome. Since DNA methylation may not be complete immediately following repair synthesis, studies are designed to determine if methylation in or near a repair batch occurs during a subsequent round of DNA replication. In other studies inhibitors of methylation (5 azacytidine and ethionine) will be used to uncouple repair synthesis and methylation and to determine if the appropriate sites can be methylated later in G1 or in a subsequent S phase. Restriction nucleases will be used to investigate whether or not here is demethylation on the contralateral strand in response to hypomethylation in or near a repair patch. Other studies are designed to examine the distribution of methylation during excision repair with respect to chromatin structure.